Method of treating hepatitis virus infections

ABSTRACT

A method of treating hepatitis virus infection is disclosed. The method comprising administering to a human subject in need of such treatment an effective hepatitis virus-combatting amount of an alkyl lipid or alkyl lipid derivative.

"The invention was made with the support of NIH Grant No. 5-RO1-CA42216.The U.S. government has certain rights in the invention".

This is a continuation-in-part application of U.S. patent applicationSer. No. 08/074,943, filed Jun. 10, 1993, now abandoned which isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to the treatment of hepatitis virusinfections, and more specifically to the treatment of hepatitis virusinfections with alkyl lipids and alkyl lipid derivatives.

BACKGROUND OF THE INVENTION

The human hepatitis B virus (HBV) is one of a family of hepadnavirusesthat cause acute and chronic liver disease, including liver cancer. Thevirus is found in the body fluids of infected persons. Recognized riskfactors for infection include blood transfusion, sexual contact,hemodialysis, shared intravenous needles, acupuncture, tissuetransplantation and the like.

The virus makes three antigenic proteins during multiplication in livercells: hepatitis B surface antigen (HBsAg), hepatitis B e antigen(HBeAg) and hepatitis B core antigen (HBcAg). These three virusantigenic proteins are important as markers for determining virusinfection, as antibodies against the virus infection are made inresponse to these virus proteins in the blood.

Currently, there are no specific antiviral agents to treat acute orchronic persistent hepatitis. An HBV vaccine is available to preventinfection, and hyperimmune gamma globulin is available for temporaryprophylaxis against developing HBV infection in persons at risk. Clearlyspecific antiviral agents are needed for treatment and control of HBVinfections in humans. Alkyl lipids and derivatives are known potentbiologic agents that effectively inhibit tumor cell growth and HIV-1multiplication. See, Marx et al., J. Med. Chem. 31:858-863 (1988), andKucera et al., AIDS Res. Human Retroviruses 6:491-501 (1990). The majorsites of action of these agents involves the plasma membrane of tumorcells, of HIV-1 infected cells and protein kinase C.

Based on the foregoing, it is an object of the present invention toprovide a new treatment method for combatting the effects of hepatitisvirus infections, and for inhibiting hepatitis virus DNA and virionproduction.

It is a second object of the present invention to provide compositionsfor carrying out the same.

SUMMARY OF THE INVENTION

These and other objects are satisfied by the present invention, which asa first aspect provides a method of treating hepatitis virus infectionin a human subject in need of such treatment. The method comprisesadministering to the human subject, in an amount effective to inhibithepatitis virus DNA replication and virion production, a compound ofFormula I: ##STR1## wherein Y is S, O, NH, NCH₃, NHC(O), or NCH₃ C(O);R₁ is unbranched or branched, saturated or unsaturated C14-C18 alkyl,alkenyl, or alkynyl;

X is a covalent bond or methylene optionally substituted with hydroxyl,C1-C20 alkyl, C1-C20 alkoxy, C1-C20 alkylthio or C1-C20 alkylamido;

and D is selected from the group consisting of moieties of Formula V orFormula VI;

wherein Formula V is

    --(PO.sub.4).sup.- --E                                     (V)

wherein E is selected from the group consisting of:

-J-N⁺ (R₂)(R₃)(R₄), wherein J is C1-C4 alkyl optionally substituted oneto three times with methyl or ethyl; and R₂, R₃, and R₄ areindependently selected from the group consisting of H and C1-C3 alkyl;and

a nucleic acid base conjugate of the Formula VII ##STR2## wherein thebase is selected from the group consisting of thymine, adenine,cytosine, guanine, hypoxanthine, uracil, 2'-3'-dideoxy-3'-thiacytidine,and 2 -aminoadenine; A is H, OH, fluorine; or N₃ ; and B is H orfluorine, or A and B together form a covalent bond;

and wherein Formula VI is

    --N.sup.+ (R.sub.5)(R.sub.6)--J--W--Z.sup.-                (VI)

wherein R₅ and R₆ are independently selected from the group consistingof H and C1-C3 alkyl;

J is as defined above;

W is --OH, or --SH; and

Z⁻ is an anion;

or a pharmaceutical salt thereof.

In particular, the method of the present invention comprisesadministering a compound of Formula I to a human subject in an amounteffective to inhibit Hepatitis A virus, Hepatitis B virus, Hepatitis Cvirus, Hepatitis D virus or Hepatitis E virus.

A second aspect of the present invention is a method of inhibiting theproduction of a hepatitis virus-induced antigen. The hepatitis virus maybe Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis Dvirus or Hepatitis E virus. The antigen may be Hepatitis B virus coreantigen or e antigen. The method comprises administering to a humansubject an effective antigen-production limiting amount of a compound ofFormula I.

A third aspect of the present invention is a method of treatinghepatitis virus infection in a human subject in need of such treatment.The method comprises administering to the human subject in an amounteffective to inhibit hepatitis virus DNA replication and virionproduction a compound of Formula II: ##STR3## wherein Y is S, O, NH,NCH₃, NHC(O), or NCH₃ C(O);

R₁ is unbranched or branched, saturated or unsaturated C14-C18 alkyl,alkenyl, or alkynyl;

X is a covalent bond or methylene optionally substituted with hydroxyl,C1-C20 alkyl, C1-C20 alkoxy, C1-C20 alkylthio or C1-C20 alkylamido;

J is C1-C4 alkyl optionally substituted one to three times with methylor ethyl; and

R₂, R₃, and R₄ are independently selected from the group consisting of Hand C1-C3 alkyl; or a pharmaceutical salt thereof. In particular, themethod of the present invention comprises administering a compound ofFormula II to a human subject in an amount effective to inhibitHepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis Dvirus or Hepatitis E virus.

A fourth aspect of the present invention is a method of inhibiting theproduction of a hepatitis virus-induced antigen. These hepatitisvirus-induced antigens are antigens that are produced as a result of thehepatitis virus infection as opposed to cellular antigens. The hepatitisvirus may be Hepatitis A virus, Hepatitis B virus, Hepatitis C virus,Hepatitis D virus or Hepatitis E virus. The antigen may be Hepatitis Bvirus core antigen or e antigen. The method comprises administering to ahuman subject an effective antigen-production inhibiting amount of acompound of Formula II.

A fifth aspect of the present invention is a method of treatinghepatitis virus infection in a human subject in need of such treatmentcomprising administering to the human subject a compound of Formula III##STR4## wherein Y is S, O, NH, NCH₃, NHC(O), or NCH₃ C(O); R₁ is anunbranched or branched, saturated or unsaturated C1-C20 alkyl, alkenyl,or alkynyl;

X is a covalent bond or methylene optionally substituted 1 or 2 timeswith hydroxyl, C1-C20 alkyl, C1-C20 alkoxy, C1-C20 alkylthio or C1-C20alkylamido;

J is C1-C4 alkyl optionally substituted one to three times with methylor ethyl;

R₂ and R₃ are independently selected from the group consisting of H andC1-C3 alkyl;

W is --OH, or --SH; and

Z⁻ is an anion.

The compound is administered in an amount effective to combat thehepatitis virus infection. In particular, the method of the presentinvention comprises administering a compound of Formula III to a humansubject in an amount effective to inhibit Hepatitis A virus, Hepatitis Bvirus, Hepatitis C virus, Hepatitis D virus or Hepatitis E virus.

A sixth aspect of the present invention is a method of inhibiting theproduction of a hepatitis virus-induced antigen. The hepatitis virus maybe Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis Dvirus or Hepatitis E virus. The antigen may be Hepatitis B virus coreantigen or e antigen. The method comprises administering to a humansubject an effective antigen-production limiting amount of a compound ofFormula III.

A seventh aspect of the present invention is a method of treatinghepatitis virus infection in a human subject in need of such treatmentcomprising administering to the human subject a compound of Formula IV.##STR5## wherein Y is S, O, NH, NCH₃, NHC(O), or NCH₃ C(O);

R₁ is an unbranched or branched, saturated or unsaturated C1-C20 alkyl,alkenyl, or alkynyl,

X is a covalent bond or methylene optionally substituted with hydroxyl,C1-C20 alkyl, C1-C20 alkoxy, C1-C20 alkylthio or C1-C20 alkylamido;

The base is selected from the group consisting of thymine, adenine,cytosine, guanine, hypoxanthine, uracil, 2'-3'-dideoxy-3'-thiacytidine,and 2-aminoadenine;

A is H, OH, fluorine, or N₃ ; and B is H or flourine, or A and Btogether form a covalent bond. In particular, the method of the presentinvention comprises administering a compound of Formula IV to a humansubject in an amount effective to inhibit Hepatitis A virus, Hepatitis Bvirus, Hepatitis C virus, Hepatitis D virus or Hepatitis E virus.

An eighth aspect of the present invention is a method of inhibiting theproduction of a hepatitis virus-induced antigen. The hepatitis virus maybe Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis Dvirus or Hepatitis E virus. The antigen may be Hepatitis B virus coreantigen or e antigen. The method comprises administering to a humansubject an effective antigen-production inhibiting amount of a compoundof Formula IV.

A ninth aspect of the present invention is the use of an alkyl lipid oralkyl lipid derivative of formulas I, II, III, or IV given above for thepreparation of a medicament combatting hepatitis virus infection andinhibiting hepatitis virus virion production. The hepatitis virus may beHepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis Dvirus or Hepatitis E virus.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 depicts biological activity of the alkyl lipid CP-51 in rat serumafter a single oral dose of the compound was administered to SpragueDawley rats.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "alkyl" is intended to refer to an unbranchedor branched alkyl group comprising carbon atoms, such as methyl, ethyl,propyl, isopropyl, n-butyl, tert-butyl, hexyl, and octyl. Thisdefinition also applies to an alkyl moiety in the alkoxy group. Examplesof alkoxy groups are methoxy, ethoxy, propoxy, and isohexoxy. Similarly,the term "alkenyl" means an unbranched or branched alkenyl groupcomprising carbon atoms and having at least one double bond, such asethenyl, propenyl, isopropenyl, n-butenyl, tert-butenyl, hexenyl, andoctenyl. The term "alkynyl" means an unbranched or branched alkynylgroup comprising carbon atoms and having at least one triple bond.

The term "pharmaceutical salt" refers to a salt that retains the desiredbiological activity of the parent compound and does not impart undesiredtoxicological effects thereto. Examples of such salts are (a) saltsformed with cations such as sodium, potassium, NH₄ ⁺, magnesium, calciumpolyamines, such as spermine, and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The term "amphipathic" refers to a compound having both a polarhydrophilic end and a non-polar hydrophobic end. The term "amphoteric"refers to a compound that has both a negative and positive charge withinthe same compound.

The present invention is directed to the use of alkyl lipids andquaternary amine containing ether lipid derivatives and therapeuticcompositions in the therapy of hepatitis virus infections. Suchinfections may be by Hepatitis A virus, Hepatitis B virus, Hepatitis Cvirus, Hepatitis D virus, or Hepatitis E virus. In particular, thepresent invention is directed to the treatment of Hepatitis B virusinfection. It has been discovered that the compounds of Formulas I, II,III and IV above can be used to treat hepatitis virus infection andvirion production in human subjects in need of such treatment, as viralhepatitis is combatted by administration of alkyl lipids or derivativesthereof to such human subjects. While the inventors do not wish to bebound by any mechanism that explains how these compounds combathepatitis virus infection, it has also been observed that production ofHepatitis B virus core and e antigens is inhibited by treatment withthese compounds. It is believed that the site of action most likelyinvolves inhibition of Hepatitis B virus DNA and post-transcriptionalprotein synthesis and replication at cell membranes.

A first aspect of the invention is a method of inhibiting hepatitisvirus DNA and hepatitis virus antigen virion production using a compoundof Formula I, wherein R₁, Y, X, and D are defined as stated above, or apharmaceutical salt thereof. The compounds of Formula I are amphipathicmoieties having a short alkyl backbone (represented by C-X-C in FormulaI), a hydrophobic end represented by R₁, linked to one end of the alkylbackbone by the functional group Y, and a hydrophilic end D linked tothe other end of the short alkyl chain. D is generally amphoteric and ispreferably a phospho-ammonium or -nucleic acid-base complex (Formula V)or an alkyl ammonium-anion complex (Formula VI).

Formulas II, III, and IV represent preferred embodiments of thecompounds of Formula I. The individual components of each are describedin detail below.

In Formula II, as described above, R₁ is a lipophilic moiety; thelipophilicity of R₁ allows the compounds of Formula II to bind with thecell membrane of a cell infected with the hepatitis virus to provide ananchor thereto. R₁ can be an unbranched or branched, saturated orunsaturated C14-C18 alkyl, alkenyl, or alkynyl.

In compounds of Formula II, Y is a functional group that links thelipophilic moiety R₁ and the short alkyl backbone of the compound. Yshould be a functional group, such as S, O, NH, NCH₃, NHC(O), or NCH₃C(O), that is able to withstand the hydrolytic activity of cellularlipases. Preferably, Y is S or NHC(O).

The alkyl backbone includes a constituent X which can be a covalent bondbetween the carbon atoms at either end of the backbone or a methyleneoptionally substituted with hydroxyl, C1-C20 alkyl, C1-C20 alkoxy,C1-C20 alkylthio or C1 -C20 alkylamido. Preferably, X is a covalent bondor a methylene substituted with a hydroxyl or C1-C4 alkoxy; morepreferably, X is methylene substituted with hydroxyl, methoxy or ethoxy.Most preferably, X is ethoxy.

The polar hydrophilic end of the amphipathic compounds of Formula IIcomprises an amphoteric ammonium phosphoalkyl group in which thephosphate moiety carries the negative charge and the ammonium moietycarries the positive charge. In the ammonium phosphoalkyl group, J isC1-C4 alkyl optionally substituted one to three times with methyl orethyl, and R₂, R₃, and R₄ are independently selected from the groupconsisting of hydrogen and C1-C3 alkyl. It is preferred that J beunsubstituted ethylene. It is also preferred that R₂, R₃, and R₄ areeach methyl. It is particularly preferred that J is unsubstitutedethylene and R₂, R₃, and R₄ are each methyl.

Exemplary compounds of Formula II include rac-3-octadecanamido-2-ethyoxy-1-propylphosphocholine (hereinafter CP-51),rac-3-hexadecanamido-2-ethoxy-1-propylphosphocholine (hereinafterCP-49), 2-hexadecylthio-1 -ethylphosphocholine (hereinafter CP-9), andrac-3-octadecyloxy-2-hydroxy-1 -propyl phosphocholine (hereinafter lysoPAF).

Compounds of Formula II can be synthesized according to knownprocedures. See, e.g., Lipids 22 (11), 775-980 (1987); exemplarysynthetic procedures are set forth below in the Examples. Among numerousnoteworthy subsequent developments are the sulfur-containingphospholipids described in U.S. Pat. No. 4,444,766 to Bosies et al., thephosphoric acid ester derivatives of 1,3 -dioxy propane disclosed inU.S. Pat. No. 4,426,525 to Hozumi et al., the cyclammonium saltsdisclosed (as platelet activating factor inhibitors) in U.S. Pat. No.4,619,917 to Lee et al., and the lipoidal amine disclosed by J. Wolff etal., Cancer Immunol. Immunother. 12:97-98 (1982).

Another aspect of the invention is the inhibition of hepatitis virus DNAand hepatitis virus antigen virion production using an alkyl lipid ofFormula III, wherein R₁, Y, X, J, R₂, R₃, W and Z are defined as statedabove, or using a pharmaceutical salt thereof. Compounds of Formula IIIare amphipathic moieties having a hydrophobic end (R₁) linked to ahydrophilic alkyl ammonium-anion complex by a short alkyl backbone,wherein the polar, hydrophilic end is an inverse choline (e.g.,N,N-dimethyl-β-hydroxyethyl ammonium).

R₁ can be an unbranched or branched, saturated or unsaturated C1-C20alkyl, alkenyl, or alkynyl. As with the compounds of Formulas I and II,R₁ is a lipophilic moiety which binds with the cell membrane of infectedcells to provide an anchor thereto. Preferably, R₁ is unbranchedsaturated or unsaturated C1-C20 alkyl. More preferably, R₁ is unbranchedsaturated or unsaturated C14-C18 alkyl.

As with the compounds of Formulas I and II, in compounds of Formula IIIY is a functional group that links the lipophilic moiety R₁ and theshort alkyl backbone of the compound. Y should be a functional group,such as S, O, NH, NCH₃, NHC(O), or NCH₃ C(O), that is able to withstandthe hydrolytic activity of cellular lipases. Preferably, Y is S orNHC(O).

The alkyl backbone of Formula III includes a constituent X which can bea covalent bond between the carbon atoms at either end of the backboneor a methylene optionally substituted with hydroxyl, C1 -C20 alkyl,C1-C20 alkoxy, C 1-C20 alkylthio or C1-C20 alkylamido. Preferably, X isa covalent bond or a methylene substituted with a hydroxyl or C1-C4alkoxy; more preferably, X is methylene substituted with hydroxyl,methoxy or ethoxy.

The polar hydrophilic end of the amphipathic compounds of Formula IIIcomprises an alkyl ammonium-anion complex wherein the anion, Z, carriesthe negative charge and the ammonium moiety carries the positive charge.In the alkyl ammonium moiety, J is C1-C4 alkyl optionally substitutedone to three times with methyl or ethyl, W is OH or SH, and R₂ and R₃are independently selected from the group consisting of hydrogen andC1-C3 alkyl. It is preferred that J is unsubstituted ethylene and W isOH. It is also preferred that R₂ and R₃ are each methyl. It is morepreferred that J is unsubstituted ethylene, W is OH, and R₂ and R₃ areeach methyl.

An exemplary compound of Formula III is N- rac-3-(hexadecylthio)-2-methoxy-1-propyl!-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide(hereinafter CP-7).

In addition to CP-7, which is synthesized according to Example 5 below,compounds of Formula III can be synthesized by following the teachingsof Example 5 in combination with procedures known to those skilled inthe art.

An additional aspect of the invention is a method of inhibitinghepatitis virus DNA and hepatitis virus antigen virion production usinga compound of Formula IV, wherein R₁, Y, X, A, B and Base are defined asstated above, or using a pharmaceutical salt thereof. Compounds ofFormula IV are amphipathic moieties wherein the polar hydrophilic end isa phospho-nucleic acid conjugate.

In the compounds of Formula IV, the non-polar hydrophobic end is R₁,which can be an unbranched or branched, saturated or unsaturated C1-C20alkyl, alkenyl, or alkynyl group. As for the compounds of Formulas I, IIand III, R₁ is a lipophilic moiety which binds with the cell membrane ofan hepatitis virus infected cell to provide an anchor for the compoundthereto. Preferably, R₁ is an unbranched saturated or unsaturated C1-C20alkyl group. More preferably, R₁ is an unbranched saturated orunsaturated C14-C18 alkyl group.

As with the compounds of Formulas I, II and III, in compounds of FormulaIV Y is a functional group that links the lipophilic moiety R₁ and theshort alkyl backbone of the compound. Y should be a functional group,such as S, O, NH, NCH₃, NHC(O), or NCH₃ C(O), that is able to withstandthe hydrolytic activity of cellular lipases. Preferably, Y is S orNHC(O).

The alkyl backbone of Formula IV includes a constituent X which can be acovalent bond between the carbon atoms at either end of the backbone ora methylene optionally substituted with hydroxyl, C1-C20 alkyl, C1-C20alkoxy, C1-C20 alkylthio or C1-C20 alkylamido. Preferably, X is acovalent bond or a methylene substituted with a hydroxyl or C1-C4alkoxy; more preferably, X is methylene substituted with hydroxyl,methoxy or ethoxy.

In the amphipathic compounds of Formula IV, the polar hydrophilic end ofthe compound comprises a phospho-nucleic acid conjugate. Many of thenucleic acid moieties that are suitable for use with the presentinvention are moieties that have shown anti-retroviral activity on otherviruses by a different mechanism than that postulated herein, and thusare attached to the compounds of Formula IV to provide an additionalimpediment to viral activity. The nucleotide base is selected from thegroup consisting of thymine, adenine, cytosine, guanine, hypoxanthine,uracil, 2'-3'-dideoxy-3'-thiacytidine, and 2-aminoadenine. Thymine is apreferred base. A is hydrogen, fluorine or N₃, and B is hydrogen orfluorine, or A and B together form a covalent bond (i.e., there is adouble bond between A and B). It is preferred that A is hydrogen or N₃and B is hydrogen. A particularly preferred nucleotide moiety is3'-azido-3'deoxythymidine (AZT) or 2'-3 '-dideoxy-3'-cytidine.

An exemplary compound of Formula IV is 3'-Azido-3'-deoxy-5'-(rac-3-dodecyloxy-2-decyloxy-1-propyl) phosphothymidine (hereinafter CP-126).

CP-126 and other compounds of Formula IV can be synthesized according tothe method of U.S. Pat. No. 5,512,627 to Piantadosi et al., the entiretyof which is herein incorporated by reference, and by the methods setforth in Example 4 below.

Experimentation has demonstrated the efficacy of the compounds ofFormulas I, II, III, and IV in combatting hepatitis virus infection (seebelow Examples 6 and 7). For example, both compounds CP-49 and CP-51substantially inhibited the levels of HBV virion DNA and intracellularHBV-DNA replication intermediate (RI) to levels comparable to, orgreater than, that observed following evaluation of an internal positivecontrol compound 2', 3'-dideoxycytidine (ddC). Compounds CP-7, CP-9 andCP-126 were moderately inhibitory of HBV replication. The levels ofvirion DNA and intracellular HBV DNA replication intermediates werereduced to amounts comparable to, but slightly less than, for ddC. Inaddition, CP-51 has been demonstrated to inhibit the production of theHBV antigens core antigen and e antigen. This result suggests that themechanism of action of the compounds involves suppression ofnucleocapsid and HBV pregenomic RNA packaging to form new HBV particles.

In the manufacture of a medicament according to the invention,hereinafter referred to as a "formulation," the compounds of Formulas I,II, III and IV are typically admixed with, among other things, anacceptable carrier. The carrier must, of course, be acceptable in thesense of being compatible with any other ingredients in the formulationand must not be deleterious to the patient. The carrier may be a solidor a liquid, or both, and is preferably formulated with the compound asa unit-dose formulation, for example, a tablet, which may contain from0.5% to 95% by weight of the active compound. One or more activecompounds may be incorporated in the formulations of the invention,which may be prepared by any of the well known techniques of pharmacyconsisting essentially of admixing the components.

The formulations of the invention include those suitable for oral,rectal, topical, intrathecal, buccal (e.g., sub-lingual), parenteral(e.g., subcutaneous, intramuscular, intradermal, or intravenous) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above).

Suitable solid diluents or carriers for the solid oral pharmaceuticaldosage unit forms are selected from the group consisting of lipids,carbohydrates, proteins and mineral solids, for example, starch,sucrose, lactose, kaolin, dicalcium phosphate, gelatin, acacia, cornsyrup, corn starch, talc and the like.

Capsules, both hard and soft, are filled with compositions of theseamino-amide active ingredients in combination with suitable diluents andexcipients, for example, edible oils, talc, calcium carbonate and thelike, and also calcium stearate.

In general, the formulations of the invention are prepared by uniformlyand intimately admixing the active compound with a liquid or finelydivided solid carrier, or both, and then, if necessary, shaping theresulting mixture. For example, a tablet may be prepared by compressingor molding a powder or granules containing the active compound,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the compound in afree-flowing form, such as a powder or granules optionally mixed with abinder, lubricant, inert diluent, and/or surface active/dispersingagent(s). Molded tablets may be made by molding, in a suitable machine,the powdered compound moistened with an inert liquid binder.

Liquid preparations for oral administration are prepared in water oraqueous vehicles which advantageously contain suspending agents, forexample, methylcellulose, acacia, polyvinylpyrrolidone, polyvinylalcohol and the like.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the active compound in a flavored base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration conveniently comprise sterile aqueous preparations of theactive compound, which preparations are preferably isotonic with theblood of the intended recipient. These preparations are preferablyadministered intravenously, although administration may also be effectedby means of subcutaneous, intramuscular, intrathecal, or intradermalinjection. The formulation should be sufficiently fluid that easysyringe ability exists. Such preparations may conveniently be preparedby admixing the compound with water or a glycine buffer and renderingthe resulting solution sterile and isotonic with the blood. Suchpreparations should be stable under the conditions of manufacture andstorage, and ordinarily contain in addition to the basic solvent orsuspending liquid, preservatives in the nature of bacteriostatic andfungistatic agents, for example, parabens, chlorobutanol, benzylalcohol, phenol, thimerosal, and the like. In many cases, it ispreferable to include osmotically active agents, for example, sugars orsodium chloride in isotonic concentrations. Injectable formulationsaccording to the invention generally contain from 0.1 to 5% w/v ofactive compound and are administered at a rate of 0.1 ml/min/kg.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by admixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include vaseline, lanoline,polyethylene glycols, alcohols, and combinations of two or more thereof.The active compound is generally present at a concentration of from 0.1to 15% w/w, for example, from 0.5 to 2% w/w.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchessuitably contain the active compound as an optionally buffered aqueoussolution of, for example, 0.1 to 0.2M concentration with respect to thesaid active compound.

Formulations suitable for transdermal administration may also bedelivered by iontophoresis (see, for example, Pharmaceutical Research 3(6), 318, (1986)) and typically take the form of an optionally bufferedaqueous solution of the active compound. Suitable formulations comprisecitrate or bistris buffer (pH 6) or ethanol/water and contain from 0.1to 0.2M active ingredient.

The compounds of Formulas I, II, III and IV are administered in anamount sufficient to inhibit hepatitis virus DNA and virion production.The dose can vary depending on the compound selected for administration,the human subject, the route of administration, and other factors.Preferably, the compound is administered in an amount of at least 0.1mg/kg, 1 mg/kg, 10 mg/kg or more, and is adminstered in an amount nogreater than 0.1 g/kg, 0.01 g/kg, 0.001 g/kg, or less.

The invention is illustrated in greater detail in the followingnon-limiting examples. In the Examples, "mg" means milligrams, "pg"means picograms, "nM" means nanomolar, "μM" means micromolar, "ml" meansmilliliters, "°C." means degrees Celsius, "DMF" means dimethylformamide,"mol" means moles, "mmol" means millimoles, and "Kg" means Kilograms.

EXAMPLE 1 Preparation of(+)-3-N-Octadecanamido-2-ethoxypropyl-1-phosphocholine

A. Preparation of (+)-3-Octadecanamido-1,2-propanediol

To a mechanically stirred solution of 3-amino-1,2-propanediol (32 g,0.35 mol) in 100 mL of pyridine and 250 mL of DMF was added a solutionof stearoyl chloride (100.0 g, 0.33 mol) in 150 mL of DMF. Afterstirring for 1 h, precipitation occurred and an additional 100 mL of DMFwas added. After 2 h, the gelatinous mass was filtered, washed withwater, and air dried. The solid was recrystallized successively fromEtOH, isopropanol, and chloroform to give 74 g (63%) as a white powder(mp 111.5°-113.5° C.). ¹ H-NMR: 0.86 (t,3H, CH₃), 1.25 (broad m, 28H,(CH₂)₁₄), 1.55 (m, 2H, NHCOCH₂ CH₂), 2.20 (t, 2H NHCOCH₂), 3.4 (m, 2H,CH₂ NH), 3.55 (d, 2H, CH₂ OH). 3.75 (m, 1H, CH), 5.8 (m, 1H, NH).

B. Preparation of (+)-3-N-Octadecanamido-1-triphenylmethyl-2-propanol

Trityl chloride (40 g, 0.11 mol) was added to a stirring solution of(+)-3 -octadecanamido-1,2-propanediol (35 g, 0.1 mol) in 250 mL ofpyridine. The reaction mixture was heated to 45°-50° C. for 10 h. Afterremoving the pyridine under reduced pressure, the residue was dilutedwith 100 mL of water and extracted three times with 100 mL ofchloroform. The combined extracts were washed with 50 mL each of cold,5% HCI and saturated NaCl, dried over sodium sulfate, filtered, andevaporated to dryness. The crude residue was recrystallized two timesfrom hexane, thereby giving 43 g (72%, mp 86°-88° C.) of the tritylether. ¹ H-NMR: 0.90 (t, 3H, CH₃), 1.25 (broad m, 28H, (CH₂)₁₄), 1.55(m, 2H, NHCOCH₂ CH₂), 2.10 (t, 2H, NHCOCH₂), 3.15 (overlapping m, 3H,CH₂ NH, CHH'OTr), 3.5 (m, 1H, CHH'OTr), 3.85 (m, 1H, CH), 5.6 (m, 1H,NH), 7.35 (m, 15H, aromatic H).

C. Preparation of (+)-3-N-Octadecanamido-2-ethoxy-1-triphenylmethoxypropane

A solution of (+)-3-N-Octadecanamido-1-triphenylmethory-2-propanol (28g, 0.045 mol) in 100 mL THF was added to a slurry of 80% NaH (1.8 g,0.05 mol) in 10 mL THF. After stirring for 30 min at room temperature,ethyl iodide (4 mL, 7.8 g, 0.05 mol) was added and the reaction mixtureheated to 50° C. for 2 h. An additional 0.3 g NaH and 2 mL of EtI wasadded and heating continued for 2 h. After cooling, water was addedslowly to decompose any residual NaH. Diethyl ether (100 mL) was addedand the layers separated. The aqueous layer was reextracted with etherand the organic extracts combined, washed with brine, and dried oversodium sulfate. The crude product was dissolved in hot hexane and asmall amount of insoluble material filtered and discarded. After coolingat 0° C., 21.7 g (78%, mp 58°-61° C.) of product was obtained.Chromatography of silica gel using hexane: ¹ H-NMR: 0.80 (t, 3H, CH₃),1.25 (broad m, 31H, (CH₂)₁₄, OCH₂ CH₃), 1.55 (m, 2H, NHCOCH₂ CH₂), 2.15(t, 2H, NHCOCH₂), 3.1.-3.7 (m, 7H, CH₂ N, CHCH₂ OH, OCH₂ CH₃), 5.7 (m,1H, NH), 7.25 (m, 15H, aromatic H).

D. Preparation of (+)-3-N-Octadecanamido-2-ethoxy-1 -propanol

p-Toluenesulfonic acid (1 g, 0.005 mol) was added to a solution of(+)-3-N-Octadecanamido-2-ethoxy-l-triphenylmethoxypropane (21.7 g, 0.035mol) in 100 mL of methylene chloride and 20 mL of methanol. The solutionwas stirred for 8 h at room temperature. Saturated sodium bicarbonatewas added and stirred for 0.5 h. The layers were separated and theorganic fraction washed with brine. After drying over sodium sulfate,the solution was concentrated in vacuo. Crude product was obtained byprecipitation from hexane. Chromatography on silica gel with a gradientof methylene chloride: methanol (100:0 to 95:5) gave 9 g (69%, mp79°-80° C.) as a white solid. ¹ H-NMR: 0.85 (t, 3H, CH₃), 1.25 (broad m,31H, (CH₂),₁₄, OCH₂ CH₃), 1.55 (m, 2H, NHCOCH₂ CH₂), 2.20 (t, 2H,NHCOCH₂), 3.3-3.9 (overlapping m, 7H, CH₂ N, CHCH₂ OH, OCH₂ CH₃), 5.9(m, 1H, NH).

E. Preparation of (+)-3-N-Octadecanamido-2-ethoxypropvl-1-phosphocholine(CP-51)

To a cooled (ice bath) stirring solution of (+)-3-N-Octadecanamido-2-ethoxy-1-propanol (1.0 g, 0.0026 mol) and triethylamine (0.29 g, 0.0029mol) in 40 mL of dry benzene was added2-chloro-2-oxo-1,3,2-dioxophospholane (0.41 g, 0.0029 mol) in 4 mLbenzene. The reaction mixture was stirred at room temperature for 4 h.The precipitate was filtered and washed with benzene. The filtrate wasconcentrated and the intermediate phosphotriester was used withoutfurther purification. The solid was transferred to a dry pressure flaskcontaining 40 mL of dry acetonitrile and cooled in a dry ice/acetonebath. Trimethylamine (3 mL) was condensed and added to the reactionvessel. The flask was sealed and the reaction mixture heated to 65° C.overnight. A white solid formed upon cooling and was filtered andprecipitated from chloroform:acetone (1:10). Chromatography on silicagel eluting with chloroform:methanol:ammonium hydroxide (70:35:2 to70:35:5) gave pure product (0.6 g, 42%, hygroscopic solid, decomposes245° C.) which was precipitated again from chloroform:acetone. ¹ H-NMR:0.85 (t, 3H, CH₃), 1.15 (t, 3H, OCH₂ CH₃), 1.25 (broad m, 28H, (CH₂ ₁₄),1.60 (m, 2H, NHCOCH₂ CH₂), 2.18 (t, 2H, NHCOCH₂), 3.35 (s, 9H, N(CH₃)₃),3.3-3.7 (overlapping m, 6H, CH₂ N(CH₃)₃, CH₂ NH, OCH₂ CH₃), 3.7-4.1 (m,3H, CH, CH₂ OP), 4.35 (m, 2H, POCH₂), 7.1 (m, 1H, NH). Elementalanalysis C₂₈ H₅₉ N₂ O₆ P•H₂ O

EXAMPLE 2 Preparation of(+)-3-N-Hexadecanamido-2-ethoxypropyl-1-phosphocholine (CP-49)

This compound was prepared by the same procedure set forth in Examples 1with the substitution of palmitoyl chloride for stearoyl chloride in thereaction described in section A above. Alternatively, this compound wasprepared using the following method. To a solution of alcohol (0.2grams, 0.5 mmol) in anhydrous diethyl ether:THF (2:1, 12 mL) at 0° C.was added dry pyridine (0.6 mL, 7 mmol) followed by 2-bromoethyldichlorophosphate (0.3 mL, 1.9 mmol). The reaction mixture was warmed toroom temperature and heated at a gentle reflux for 4 hours. The heat wasremoved, water (3 mL) was added and the reaction mixture stirred for 0.5hours. Solvent was removed and the residue dissolved inchloroform:methanol (2:1). This solution was extracted with water, driedover sodium sulfate, and the solvent removed on a rotary evaporator.Column chromatography on silica gel with a gradient of chloroformmethanol (100:0 to 3:1) was used to purify the bromoethyl phosphate(0.15 grams, 52%, R₁ -0.3 in chloroform:methanol 4:1). ¹ H--NMR: 0.85(t, 3H, CH₃), 1.15 (t, 3H, OCH₂ CH₃), 1.25 (broad m, 28H, (CH₂)₁₄), 1.6(m, 2H, NHCOCH₂ CH₂), 2.28 (t, 2H, NHCOCH₂), 3.3-4.0 (overlapping m, 9H,CH₂ Br, CH₂ NH, OCH₂ CH₃, CHCH₂ OP), 4.2 (m, 2H, POCH₂) 6.65 (m, 1H,NH). Proton nuclear magnetic resonance spectra were recorded in CDCl₃ oneither a Bruker 300-MHz or a Varian 400-MHz spectrometer. Chemicalshifts are reported in parts per million relative to internaltetramethylsilane. The bromoethyl phosphate intermediate was dissolvedin a solution of chloroform:isopropanol:DMF (3:5:5) and trimethylamine(40% aqueous, 2 mL) was added. The reaction mixture was heated to 65° C.for 5 hours and then allowed to cool to room temperature before silvercarbonate (90 mg) was added. Heat was reapplied for 1 hour, then thereaction mixture was cooled and filtered. Solvent was removed in vacuoand the phospholipid purified by column chromatography giving 68 mg(47%). Alternatively, the displacement of bromide can be performed withcondensed trimethylamine gas in acetone in a heated pressure flask.

EXAMPLE 3 Preparation of 2-(Hexadecylthio)ethyl phosphocholine (CP-9)

A. Preparation of 2-(Hexadecylthio)ethanol

Thioethanol (5.0 g, 64 mmol), hexadecyl bromide (25.0 g, 82 mmol), andKOH (4.5 g, 80 mmol) were combined in 95% EtOH (150 mL). The reactionmixture was stirred at room temperature overnight and then diluted withH₂ O. The precipitate was collected and recrystallized from MeOH toprovide the thioether: 19.0 g, 96%; mp 50° C.; NMR (300 MHz, CDCl₃)δ0.89(t, 3 H, CH₃), 1,30 (m, 26 H, (CH₂)₁₃), 1.60 (m, 2 H, CH₂ CH₂ S), 2.52(t, 2 H, SCH₂), 2.72 (m, 2 H, CH₂ S), 3.72 (m, 2 H, CH₂ OH).

B. Preparation of 2-(Hexadecylthio) ethylphosphocholine (CP-9)

2-(Hexadecylthio)ethanol (1.0 g, 3.0 mmol) and Et₃ N (0.40 g, 4.0 mmol)were dissolved in anhydrous benzene (75 mL). The solution was cooled to0° C. before a solution of 2-chloro-2-oxo-1,3,2-dioxaphospholane (0.65g, 4.6 mmol, Fluka) in anhydrous benzene was slowly added. The reactionmixture was stirred overnight at room temperature and then filtered. Thefiltrate was reduced, and the residue was dissolved in CH₃ CN (50 ml)and transferred to a glass bomb. Condensed N(CH₃)₃ (2.0 g, 34 mmol) wasadded, and the mixture was heated at a gentle reflux for 24 h. Uponcooling of the reaction mixture, a white precipitate formed. The solidwas removed and recrystallized with Et₂ O to provide 980 mg of2-(Hexadecylthio)ethylphosphocholine. 70%; dec >200° C.; NMR (400 MHz,CDCl₃)δ6 0.86 (t, 3 H, CH₃), 1.21 (s, 26 H, (CH₂)₁₃), 1.53 (m, 2 H, CH₂CH₂ S), 2.51 (t, 2 H, SCH₂), 2.72 (t, 2 H, CH₂ S), 3.37 (s, 9 H,N(CH₃)₃), 3.81 (m CH₂ N), 3.91 (m, 2 H, CH₂ OP), 4.31 (m, 2 H, POCH₂);FAB MS m/e 468 (MH+).

EXAMPLE 4 Preparation of3'-Azido-3'-deoxy-5'-(3-dodecyloxy-2-decyloxypropyl)-phosphothymidine(CP-126)

A. Preparation of 3-Dodecyloxy-1 2-propanediol

Isopropylideneglycerol (solketal, 26.4 g, 0.20 mol) in 60 mL of toluenewas added dropwise to a solution of powered KOH (22.4 g, 0.04 mol) in150 mL of toluene. The resulting mixture was refluxed for 4 h.1-Bromododecane (50 g, 0.20 mol) in 40 mL of toluene was then addeddropwise, and the solution was refluxed for 10 h. After cooling, thereaction mixture was diluted with 200 mL of ice-water and extracted withdiethyl ether (3×100 mL). The ether layers were dried over magnesiumsulfate, and the solvent was removed in vacuo. The residue was dissolvedin 60 mL of diethyl ether and 260 mL of MeOH. Concentrated HCl (60 mL)was added, and the solution refluxed for 16 h. After cooling, ice-water(150 mL) was added, and the layers separated. The aqueous layer wasextracted with diethyl ether (2×75 mL). The combined organic fractionswere then dried over sodium sulfate, filtered, and concentrated invacuo. The solid residue was recrystallized from MeOH to give 37 g (0.14mol, 71%) of a white solid.

B. Preparation of 3-Dodecyloxy-1 -triphenylmethory-2-propanol

The diol produced in section A was tritylated with trityl chloride (59g, 0.21 mol) in pyridine (200 mL) at 70° C. for 5 h and then at roomtemperature overnight. The pyridine was removed under vacuum, and thesolid residue partitioned between water and CHCl₃. The CHCl₃ layer waswashed with 5% HCI and water, then dried over magnesium sulfate. Afterremoval of solvent, the product was recrystallized from hexanes:ethylacetate (10:1) to give 19 g of pure 3-dodecyloxy-1-triphenylmethoxy-2-propanol.

C. Preparation of 3-Dodecyloxy-2-decyloxy-1-triphenylmethoxypropane

3-Dodecyloxy-1-triphenylmethoxy-2-propanol (13.5 g, 0.027 mol) was addeddropwise to an ice-cooled suspension of sodium hydride (80%, 1.6 g,0.054 mol) in 150 mL of tetrahydrofuran under nitrogen. After stirringfor 2 h at room temperature, heat was applied (55° C.). 1-Bromodecane (6g, 0.027 mol) was added dropwise, and heating continued for 6 h. Aftercooling for 3 h, water was added slowly. Diethyl ether (2×100 mL) wasadded, and the solution washed with 15% sodium thiosulfite, water, andbrine. After drying over sodium sulfate, the ether was removed, and theresidue chromatographed with a gradient of hexanes:ethyl acetate (100:0to 20:1) to give 9 g (52%) of a clear liquid.

D. Preparation of 3-Dodecyloxy-2-decyloxy-1-propanol

Detritylation of 3-dodecyloxy-2-decyloxy-1 -triphenylmethoxypropane wasaccomplished using p-toluenesulfonic acid (0.9 g) in CHCl₃ :MeOH (72mL:36 mL) (stirred at room temperature for 48 h, added 10% sodiumbicarbonate, extracted with CHCl₃, dried over magnesium sulfate, andconcentrated). The residue was purified by column chromatography using agradient of hexanes:ethyl acetate (20:1 to 5:1) to give 3.5 g (63%) ofpure 3-dodecyloxy-2-decyloxy-1-propanol.

E. Preparation of 3-Dodecyloxy-2-decyloxypropyl Diphenyl Phosphate

Diphenylchlorophosphate (0.7 mL, 3.4 mmol) in 10 mL of diethyl ether wascooled to 4° C. under nitrogen. 3-Dodecyloxy-2-decyloxy-1-propanol (1.0g, 2.6 mmol) in 15 mL of pyridine and 5 mL of diethyl ether was added.The solution was warmed to room temperature, then heated to 52° C. for 3h. It was then cooled to room temperature, diluted with 50 mL of diethylether, washed with water (2×25 mL), 0.5N HCl (25 mL), and finally withwater (25 mL). The organic layer was dried over sodium sulfate,filtered, and concentrated in vacuo to an oil. Chromatography with agradient of hexanes:ethyl acetate (10:1 to 1:1) gave 980 mg (1.5 mmol,60%) of pure product.

F. Preparation of 3-Dodecyloxy-2-decyloxypropyl Phosphate

PtO₂ (69 mg) was placed in a Parr hydrogenation bottle.3-Dodecyloxy-2-decyloxypropyl Diphenyl Phosphate (500 mg) in 100 mL ofEtOH was then added. The reaction mixture was hydrogenated at 15 psi for1.5 h until hydrogen uptake stopped. The reaction mixture was thenfiltered through Celite, and the EtOH removed in vacuo. The oil residuewas dissolved in 25 mL of pyridine, concentrated in vacuo, and driedunder high vacuum to give 350 mg of pure solid phosphatidic acid.

G. Preparation of3'-Azido-3'-deoxy-5'-(3-dodecyloxy-2-decyloxypropyl)-phosphothymidine(CP-126)

AZT (43 mg, 0.16 mmol) and 3-dodecyloxy-2-decyloxypropyl phosphate (105mg, 0.22 mmol) were azeotrophically dried with pyridine (3×3 mL) by invacuo removal. Dicyclohexylcarbodiimide (220 mg, 1.07 mmol) was addedand the drying repeated 4 times. A final 3 mL portion of pyridine wasadded, and the reaction mixture stirred at room temperature in adesiccator for 4 days. Water (1 g) was added, and the mixture stirredfor 4 h. The solvents were removed in vacuo, and the crude materialchromatographed on 2 g of silica gel using a gradient of CHCl₃ :MeOH(15:1 to 2:1). The product was dissolved in 11 mL of CHCl₃ :MeOH:H₂ O(4:6:1) and stirred with 1.5 g of Whatman preswollen microgranularcation (Na+) exchange (carboxymethyl)-cellulose resin for 1 h to obtainthe sodium salt. The resin was filtered and concentrated in vacuo togive 37 mgof3'-Azido-3'deoxy-5'(3-dodecyloxy-2-decyloxypropyl)-phosphothymidine (CP-126)(22%). FAB ms showed a MH+Na!+ ion at 752.4350 (C₃₅ H₆₄ N₅ O₉ PNa, 1.4ppm) and a M+2Na!+ ion at 774.4179 (C₃₅ H₆₃ N₅ O₉ PNa₂,2.0 ppm).

EXAMPLE 5 Synthesis of N-rac-3-(hexadecylthio-2-methoxy-1-propyl!-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (CP-7)

Into a two neck 25 ml round bottom flask equipped with an air condenser,thermometer and stir bar, was placed 2.0 g (0.005 mol) of(+)-1-hexadecylthio-2-methoxy-3-bromopropane, 0.5 ml (0.006 mol) ofN,N-dimethylaminoethanol and 15 ml of DMF. The solution was maintainedat 45°-50° C. for 72 hours with continuous stirring. The reactionmixture was then cooled to room temperature, 125 ml of ether was addedand the solution was kept at 0° C. for 24 hours. The resultingprecipitate (800 mg) was filtered and swirled with five 50 ml portionsofetherto give(±)-3-hexadecylthio-2-methoxy-N,N-dimethyl-N-β-hydroxyethyl-1 -propylammonium bromide (32%), (mp 107°-109° C.). ¹ H-NMR (CDCl₃): delta, 0.87(t, 3 H, terminal methyl), 1.2-1.6 m, 28 H, (CH₂)₁₄ !,2.45-3.0(m, 4 H,S-C₂, CH₂ -S), 3.48 s, 9 H, CH₃ -O, N(CH₃)₂ !, 3.9-4.3(m, 7 H, CH, CH₂N, N-CH₂ -CH₂ -OH). Anal. (C₂₄ H₅₂ NO₂ SBr) C,H,N.

EXAMPLE 6 Evaluation of Alkyl lipids for Anti-HBV Activity in HEP-G2Cells

The structures of the alkyl lipids evaluated for anti-HBV activity arepresented below: ##STR6## where R₁, R₂, and R₃ are defined below foreach identified compound.

    ______________________________________                                        Compound R.sub.1      R.sub.2   R.sub.3                                       ______________________________________                                        CP-7     SC.sub.16 H.sub.33                                                                         CH.sub.3  N.sup.+ (CH.sub.3).sub.2 (CH.sub.2).sub.2                                     OH                                            CP-48    NHC(O)C.sub.15 H.sub.31                                                                    CH.sub.3  PC                                            CP-49    NHC(O)C.sub.15 H.sub.31                                                                    CH.sub.3 CH.sub.2                                                                       PC                                            CP-50    NHC(O)C.sub.17 H.sub.35                                                                    CH.sub.3  PC                                            CP-51    NHC(O)C.sub.17 H.sub.35                                                                    CH.sub.3 CH.sub.2                                                                       PC                                            ______________________________________                                         PC = phosphocholine  OPO.sub.3 CH.sub.2 CH.sub.2 N.sup.+ (CH.sub.3).sub.3

A. Evaluation of Cytotoxicity of Alkyl Lipid Compounds

Initially, evaluations of the alkyl lipids were done to determinewhether the compounds were cytotoxic to the cells at concentrationshaving anti-HBV activity. The assay methodology is set forth in Korbaand Gerin, Antiviral Res., 19, 55-70 (1992), incorporated by referenceherein. Briefly, these cell cytotoxicity evaluations were done in96-well plates seeded with HEP-G2 cell monolayers. The hepatoblastomacell line HEP-G2 transfected with a plasmid carrying the gene thatconfers resistance to G418 and four 5'-3' tandem copies of the HepatitisB virus genome was obtained from Dr. George Acs. Proc. Natl. Acad. Sci.U.S.A., 84, 1005-1009 (1987). Serial concentrations of compound wereadded to the RPMI-1640 growth medium and the growth medium was added tothe 96-well plates containing the cell monolayers. The cells wereincubated for a total of nine days at 37° C. The compound-containingmedium was periodically replaced. Each compound was tested at fourserial concentrations in triplicate. After nine days of treatment andincubation, 0.01% neutral red dye (Sigma, Inc.) was added to the overlaymedium. Cell cytotoxicity was quantitated by measuring the average A₅₁₀. Values in Table I represent the percentage of average A₅₁₀ values(±standard deviation) relative to nine non-treated control wells.

                  TABLE 1                                                         ______________________________________                                        EVALUATION OF ALKYL LIPIDS FOR CELL CYTOTOXICITY IN                           HEP-G2 CELLS                                                                           % of Control Neutral Red Dye Uptake at                                        Indicated Compound Conc. (μM)                                     Compound   100      30        10     3                                        ______________________________________                                        CP-7       24 ± 2                                                                              67 ± 3 102 ± 6                                                                            98 ± 2                               CP-48      24 ± 1                                                                              61 ± 2  97 ± 1                                                                           102 ± 1                               CP-49      29 ± 1                                                                              60 ± 4  99 ± 4                                                                            99 ± 3                               CP-50      95 ± 2                                                                              100 ± 3                                                                               99 ± 2                                                                            97 ± 2                               CP-51      71 ± 1                                                                              96 ± 2 100 ± 4                                                                           101 ± 1                               Control    35 ± 2                                                                              99 ± 2 102 ± 1                                                                           100 ± 1                               2',3'-dideoxycytidine                                                         Control    100 ± 1                                                                             100 ± 2                                                                              100 ± 4                                      No Compound                                                                   ______________________________________                                    

These results indicated that at a concentration of 30 μM, compoundsCP-7, CP-48, and CP-49 inhibited neutral red dye uptake by 33, 39 and40%, respectively, compared to 0 and 4% inhibition for CP-50 and CP-51,respectively. At a concentration of 100 μM , CP-50 and CP-51 inhibitedneutral red dye uptake by 5 and 29 %, respectively. These data indicatethat at anti-HBV concentrations of 10 μM (see Tables 2, 3, or 4), thecompounds did not inhibit neutral red dye uptake. In summary, thephospholipids were not cytotoxic to the cells at concentrations thathave anti-HBV activity.

B. Evaluation of Alkyl Lipids for Anti-hepatitis B Virus Activity inHEP-G2 Cells Persistently Infected with HBV

HEP-G2 cells persistently infected with HBV were treated daily withfresh D-MEM containing 20% FBS and 10 μM compound. After 9 daysincubation and treatment, the overlay medium was harvested to assay thequantity of Hepatitis B virus s antigen (HBsAg) and HBeAg by EIA (AbbottLaboratories). The overlay medium was diluted to levels of antigen inthe linear range of the assay. Standard curves using dilutions ofpositive HBsAg and HBeAg controls were included in each assay. VirionDNA was assayed using the method set forth in Korba and Gerin, AntiviralRes., 19, 55-70 (1992), incorporated by reference herein. HBV-DNA wasextracted from the overlay medium containing virus particles andanalyzed by a slot-blot hybridization technique using ³² P-labelled 3.2Kb Eco R1 HBV DNA fragment by comparisons to HBV standards present onnitrocellulose filters using an AMBIS beta scanner. EC₉₀ values (90%effective concentration, i.e., drug concentration which induces a 90%decrease in the levels of HBV DNA replication intermediates in treatedversus control cells) were calculated by linear regression analysis. Therelative levels of HBsAg, HBeAg and virion DNA were calulated as apercentage of the average levels obtained from the untreated controls.

Results from these bioassays are shown in Table 2. These resultsindicate that CP-7 had moderate anti-HBV inhibitory activity and thatCP-49 and CP-51 had potent inhibitory activity against HBV-inducedvirion DNA and HBeAg syntheses. In contrast, CP-48 and CP-50 had nosignificant activity against HBV-induced virion DNA and HBeAg syntheses.The compounds had no significant activity against HBV induced HBsAgsynthesis.

                  TABLE 2                                                         ______________________________________                                        EVALUATION OF ALKYL LIPIDS FOR ANTI-HEPATITIS B                               VIRUS ACTIVITY IN HEP-G2 CELLS PERSISTENTLY                                   INFECTED WITH HBV                                                                         Relative Level of HBV Antigens or Virion                                      DNA After 9 Days Treatment                                        Compound      (% Untreated Control)                                           (10 μM)    HBsAg       HBeAg   DNA                                         ______________________________________                                        CP-7          74          43      10                                          CP-48         73          88      96                                          CP-49         115         30      1                                           CP-50         104         93      88                                          CP-51         99          28      2                                           Control       106         108     2                                           2',3'-dideoxycytidine                                                         Control - No  98          105     97                                          Compound                                                                      ______________________________________                                    

C. Evaluation of CP-51 Against HBV-Induced Antigen Syntheses

Further studies were performed to evaluate the most potent anti-HBVcompound, CP-5 1, against HBV-induced antigen syntheses. The resultsfrom these studies are given in Table 3. The results indicate that CP-51had no inhibitory activity against HBsAg synthesis. In contrast, CP-51inhibited HBeAg and HBcAg syntheses by 76 and 68%, respectively. Thecontrol compound ddC had no inhibitory activity against HBV-inducedantigen syntheses. In summary, these results indicate that CP-51 hasselective inhibitory activity against HBeAg and HBcAg syntheses.

                  TABLE 3                                                         ______________________________________                                        EVALUATION OF ALKYL LIPIDS FOR ANTI-HEPATITIS B                               VIRUS ACTIVITY IN HEP-G2 CELLS PERSISTENTLY                                   INFECTED WITH HBV                                                                          Relative Levels of HBV Antigens                                               After 9 Days Treatment                                           Compound       (% Untreated Control)                                          (10 μM)     HBsAg      HBeAg   HBcAg                                       ______________________________________                                        CP-51          88         24      32                                          Control        96         95      102                                         2',3'-dideoxycytidine                                                         Control - No compound                                                                        100        100     100                                         ______________________________________                                         HBsAg = HBV surface antigen                                                   HBeAg = HBV "e" antigen                                                       HBcAg = HBV core antigen                                                 

D. Evaluation of Effects of CP-5 1 on HBV-Induced DNA and RNA SynthesisExperiments were performed to determine the effects of CP-51 onHBV-induced DNA and RNA syntheses. HEP-G2 cells persistently infectedwith HBV were treated daily with fresh D-MEM containing 20% FBS and 10μMcompound. After 9 days incubation and treatment the cells and overlaymedium were harvested separately to assay the quantity of nucleic acids.Details of the methodology of the assays are given in Section B above.Extracellular virion DNA in untreated cells ranged from 50 to 150 pg/mlin the overlay medium. Intracellular HBV DNA replication intermediates(RI) in untreated cells ranged from 50 to 100 pg/μg cell DNA.Hybridization analysis results in about 1 pg intracellular HBV DNA/μgcellular DNA to 2-3 genomic copies per cell and 1.0 pg of extracellularHBV DNA/ml overlay medium to 3×10³ viral particles/ml. HBV RNA wasanalyzed by Northern blot hybridization analysis using a ³² P-labeled3.2 Kb gel-purified cloned genomic probe. Quantitive analysis ofintracellular HBV DNA and HBV RNA was performed using an AMBIS betascanner.

Results presented in Table 4 indicate that CP-51 inhibited virion,replication intermediates (RI) and 3.2Kb DNA syntheses by 98, 88 and58%, respectively. In contrast, CP-51 had no significant effects onHBV-induced RNA syntheses. The control compound ddC also inhibitedHBV-induced DNA synthesis but not RNA syntheses.

                  TABLE 4                                                         ______________________________________                                        EVALUATION OF ALKYL LIPID CP-51 FOR ANTI-HEPATITIS B                          VIRUS ACTIVITY IN HEP-G2 CELLS PERSISTENTLY                                   INFECTED WITH HBV                                                                      Relative Levels of HBV Nucleic Acids After 9 Days                             Treatment (% Untreated Control)                                      Compound   Virion  HBV    3.2 Kb                                                                              Total                                                                              3.6 Kb                                                                              2.1 Kb                             (10 μM) DNA     RI     DNA   RNA  RNA   RNA                                ______________________________________                                        CP-51      2       12     42    112  84    112                                Control    2       6      46    92   92    105                                2',3'-                                                                        dideoxycytidine                                                               Control -  100     100    100   100  100   100                                No Compound                                                                   ______________________________________                                    

In summary, of the phospholipid compounds evaluated, CP-51 has the mostpotent anti-HBV activity. CP-51 is selective for inhibiting HBV-inducedHBcAg, HBeAg, virion DNA and HBV RI syntheses but not HBsAg or RNAsyntheses. These data also indicate that the antiviral activity of CP-51is not due to cell cytotoxicity of the compound.

EXAMPLE 7 Evaluation of Alkyl Lipids for Anti-HBV Activity in HBVPersistently Infected Human Hepatablastoma Cell Line HEP-G2

A. Evaluation of Cytotoxicity of Alkyl Lipid Compounds

An analysis was performed to determine the cytotoxicity of the followingcompounds in human HEP-G2 cells persistently infected with HBV: CP-51,CP-49, CP-9, CP-126, lyso PAF and CP-7. 2'-3'-Dideoxycytidine (ddC) wasused as a control in this cytotoxicity analysis. The toxicity analyseswere performed in 96-well flat bottomed tissue culture plates followingthe same procedure set forth in Example 6 above. Each compound wastested at four concentrations, each in triplicate cultures. Uptake ofneutral red dye was used to determine the relative level of toxicity.The absorbance of internalized dye at 510 nM (A₅₁₀) was used for thequantitative analysis. The results are given in Table 5.

Values are presented as a percentage of the average A₅₁₀ values(±standard deviations) in nine separate cultures of untreated cellsmaintained on the same 96-well plate as the test compounds. Thepercentage of dye uptake in the nine control cultures was 100 ±1. Celltoxicity (greater than 50% depression of the dye uptake levels observedin untreated cells) did not occur at concentrations of alkyl lipids(CP-49, CP-51, CP-7, CP-9 and CP-126) that have strong or moderateantiviral activity.

                  TABLE 5                                                         ______________________________________                                        CYTOTOXICITY ANALYSIS OF COMPOUNDS IN HBV                                     PERSISTENTLY INFECTED HUMAN HEP G2 CELLS                                                 % of Control Neutral Red Dye Uptake At                                        Indicated Compound Concentration                                   Trials                                                                             Compound    (% of Control + S.D.)                                        ______________________________________                                        1    Control     300 μM                                                                              100 μM                                                                            30 μM                                                                             10 μM                                   2'-3'-      (34 ± 4)                                                                            (97 ± 2)                                                                          (100 ± 1)                                                                         (101 ± 2)                               Dideoxycytidine                                                          1    Alkyl Lipid 100 μM                                                                              30 μM                                                                             10 μM                                                                              3 μM                                   CP-51       (85 ± 2)                                                                            (100 ± 3)                                                                         (102 ± 1)                                                                          (98 ± 3)                          2    Control     1000 μM                                                                             300 μM                                                                            30 μM                                                                             10 μM                                   2'-3'-      (12 ± 5)                                                                            (40 ± 4)                                                                          (102 ± 2)                                                                         (101 ± 3)                               Dideoxycytidine                                                          2    Alkyl Lipid 100 μM                                                                              30 μM                                                                             10 μM                                                                             30 μM                                   CP-51       (44 ± 2)                                                                            (91 ± 1)                                                                          (98 ± 4)                                                                          (100 ± 2)                          2    Alkyl Lipid 100 μM                                                                              30 μM                                                                             10 μM                                                                             30 μM                                   CP-49       (45 ± 4)                                                                            (75 ± 1)                                                                          (97 ± 2)                                                                          (100 ± 1)                          2    Alkyl Lipid 100 μM                                                                              30 μM                                                                             10 μM                                                                              3 μM                                   CP-9        (45 ± 2)                                                                            (86 ± 2)                                                                          (99 ± 2)                                                                          (100 ± 1)                          2    Alkyl Lipid-                                                                              100 μM                                                                              30 μM                                                                             10 μM                                                                              3 μM                                   AZT         (20 ± 2)                                                                            (68 ± 1)                                                                          (97 ± 3)                                                                          (100 ± 1)                               Conjugate                                                                     CP-126                                                                   2    Platelet    100 μM                                                                              30 μM                                                                             10 μM                                                                              3 μM                                   Activating  (40 ± 6)                                                                            (98 ± 2)                                                                          (99 ± 1)                                                                           (98 ± 2)                               Factor                                                                        (lyso PAF)                                                               3    Control     300 μM                                                                              100 μM                                                                            30 μM                                                                             10 μM                                   2'-3'-      (35 ± 2)                                                                            (99 ± 2)                                                                          (102 ± 1)                                                                         (100 ± 1)                               Dideoxycytidine                                                          3    Alkyl Lipid 100 μM                                                                              30 μM                                                                             10 μM                                                                              3 μM                                   CP-51       (71 ± 1)                                                                            (96 ± 2)                                                                          (100 ± 4)                                                                         (101 ± 1)                          3    Alkyl Lipid 100 μM                                                                              30 μM                                                                             10 μM                                                                              3 μM                                   CP-49       (29 ± 1)                                                                            (60 ± 4)                                                                          (99 ± 4)                                                                           (99 ± 3)                          3    Nonphosphorus                                                                             100 μM                                                                              30 μM                                                                             10 μM                                                                              3 μM                                   Alkyl Lipid (24 ± 2)                                                                            (67 ± 3)                                                                          (102 ± 6)                                                                          (98 ± 2)                               (Inverse                                                                      Choline) CP-7                                                            ______________________________________                                    

B. Evaluation of Effect of Alkyl Lipids on HBV Replication

Cells were treated with a particular compound for nine days at 37° C.Analysis of intracellular HBV DNA was done 24 hours following the ninthday of treatment. Table 6 shows the level of inhibition of HBV virionDNA and intracellular HBV DNA replication intermediates. The levels ofintegrated HBV DNA in each cellular DNA preparation were used tocalculate the levels of episomal 3.2-3.8 Kb HBV genomes (Mono) and HBVDNA replication intermediates (RI). A zero value indicates anundetectable level of HBV virion DNA. The sensitivity cut-off was 0.1 pgDNA/ml of culture medium.

                  TABLE 6                                                         ______________________________________                                        EFFECT OF ALKYL LIPIDS ON HBV REPLICATION                                     IN HBV PERSISTENTLY INFECTED HUMAN HEP G2 CELL                                                Intra-                                                                        cellular                                                                      HBV                                                                           DNA                                                                           (pg/μg                                                                     Cell     HBV Virion DNA                                                  Conc DNA)     (pg/ml Culture Medium)                               Compound     (μM)                                                                              Mono   RI  Day 0 3    6   9                               ______________________________________                                        Positive Control                                                                           10     1.0    8   67    49   20  2                               Dideoxycytidine     -1.4   8   64    55   18  3                                                   1.3    7   58    50   17  1                                                   1.8    9   59    58   15  2                               Negative Control                                                                           0      2.2    70  63    63   84  94                              Untreated           2.0    80  80    70   65  83                                                  2.6    72  66    68   70  84                                                  2.7    55  62    60   82  78                              Alkyl        10     2.6    9   73    49   21  1                               Lipid (CP-49)       2.4    8   53    55   22  1                                                   2.2    6   56    57   19  3                                                   2.7    9   77    50   24  2                                            1      2.3    24  56    56   38  10                                                  2.4    20  51    58   41  13                                                  2.2    25  66    60   43  11                                                  2.1    30  79    68   44  18                                           0.1    2.2    60  71    72   62  75                                                  2.3    61  55    56   69  65                                                  2.5    74  72    88   55  77                                                  2.6    79  62    75   55  57                              Nonphosphorus                                                                              10     2.3    11  49    70   35  8                               Alkyl Lipid                                                                   (Inverse Choline)   2.3    13  63    64   28  9                               (CP-7) C.sub.24 H.sub.52 NO.sub.2 SBN                                                             2.6    10  70    67   33  7                               N- rac-3-           2.5    14  65    59   33  7                               (hexadecylthio)-                                                              2-methoxy-1-propyl!-                                                          N,N-dimethyl-N-                                                                            1      2.7    70  53    56   46  48                              (2-hydroxyethyl)    2.2    75  64    51   52  44                              ammonium                                                                      bromide             2.3    63  54    66   59  49                                                  2.4    68  54    58   59  41                                           0.1    2.5    73  51    72   58  57                                                  2.6    62  51    75   65  89                                                  2.8    57  77    66   98  84                                                  2.3    76  64    51   75  76                              Alkyl        10     2.3    13  60    52   33  10                              Lipid (CP-9)        2.2    14  66    68   40  14                              C.sub.23 H.sub.50 NO.sub.4 SP                                                                     2.2    17  61    57   39  11                              2-Hexadecylthio-1-  2.3    12  52    60   41  14                              ethylphosphocholine                                                                        1      2.1    53  59    66   57  69                                                  2.6    82  53    60   52  71                                                  2.3    67  60    82   88  94                                                  2.6    60  58    55   78  76                                           0.1    2.0    53  50    52   83  94                                                  2.2    54  55    64   78  69                                                  2.4    64  65    90   91  80                                                  2.4    66  59    63   64  92                              Alkyl Lipid-AZT                                                                            10     1.9    14  60    59   20  13                              Conjugate           2.5    9   62    50   21  8                               (CP-126)                                                                      C.sub.35 H.sub.63 N.sub.5 O.sub.9 P                                                               2.0    12  59    55   26  9                               3'-Azido-3'-Deoxy-  2.3    16  51    66   29  12                              5'-(rac-3-                                                                    dodecyloxy-                                                                   2-decyloxy-1-propyl                                                                        1      2.3    51  67    49   40  28                              phosphothymidine    2.7    67  54    57   39  20                                                  2.1    56  70    65   44  22                                                  1.8    61  69    53   48  26                                           0.1    2.2    64  50    77   61  78                                                  2.2    54  52    79   72  64                                                  2.6    70  54    67   83  82                                                  2.4    89  60    65   66  77                              Platelet Activity                                                                          10     2.0    15  52    65   36  14                              Factor (lyso PAF)   2.4    22  62    71   34  18                              C.sub.26 H.sub.56 NO.sub.6 P                                                                      2.6    18  60    51   30  11                              rac-3-octadecyloxy- 2.8    21  68    50   32  13                              2-hydroxy-1-propyl                                                            phosphocholine                                                                             1      2.6    74  52    69   72  74                                                  2.9    83  51    61   79  83                                                  2.4    59  63    53   81  59                                                  2.6    84  65    69   79  88                                           0.1    2.5    84  62    77   80  84                                                  2.1    96  60    81   79  96                                                  2.0    67  81    70   64  67                                                  2.0    71  57    69   88  92                              Alkyl Lipid  10     1.7    4   59    55   20  2                               (CP-51)             1.9    3   58    49   13  2                                                   1.8    5   60    44   10  1                               BXP 1               2.0    6   78    48   18  1                                            1      2.2    12  66    59   29  9                                                   2.3    12  89    47   35  8                                                   2.3    16  74    59   36  12                                                  2.4    17  70    68   30  10                                           0.1    2.7    63  58    58   79  83                                                  2.4    72  55    65   57  86                                                  2.3    84  60    68   81  76                                                  2.5    78  55    79   84  80                              Alkyl        10     1.3    7   51    33   12  0                               Lipid               1.1    3   56    30   11  1                               (CP-51)                                                                                           1.2    5   58    36   10  1                               EXP2                1.0    4   64    40   14  0                                            1      2.3    14  51    59   30  9                                                   2.6    17  50    63   20  13                                                  2.0    16  57    51   33  11                                                  2.0    13  60    60   29  10                                           0.1    2.7    78  52    69   78  38                                                  2.3    60  56    66   63  40                                                  2.9    54  43    66   68  40                                                  2.2    80  59    70   70  32                              Alkyl        1      2.2    17  92    110  32  14                              Lipid               2.0    23  55    66   37  16                              (CP-51)                                                                                           2.3    21  54    69   30  12                                                  2.1    18  59    58   38  11                              EXP3         0.1    2.4    40  75    79   78  110                                                 2.6    62  67    100  85  95                                                  2.1    81  57    66   58  64                                                  2.3    51  83    62   78  49                              ______________________________________                                    

Compounds CP-49 and CP-51 markedly inhibited the levels of HBV virionDNA and intracellular HBV RI DNA to levels comparable to, or greaterthan, that observed following evaluation of an internal positive controlcompound 2',3'-dideoxycytidine (ddC). Compounds CP-7, CP-9, and CP-126were moderately inhibitory of HBV replication. The levels of virion DNAand HBV RI DNA were reduced to amounts comparable to, but slightly lessthan, the internal standard compound ddC.

C. Effect of Alkyl Lipids on Antigen Markers

Cells were treated with compound for nine days at 37° C. The overlaymedium was harvested to measure levels of HBsAg, HBeAg and virion DNA.HBsAg and HBeAg were assayed by EIA (Abbott Laboratories). Virion DNAlevels were measured as described in section B above. None of thecompounds tested (CP-51, CP-49, CP-9, CP-7 and CP-126) affectedHBV-induced hepatitis B virus surface antigen (HBsAg) levels released bypersistently infected cells into the culture medium, as shown in Table7.

                  TABLE 7                                                         ______________________________________                                        EFFECT OF ALKYL LIPIDS AND ALKYL LIPID-AZT CONJUGATE                          ON SYNTHESIS OF ANTIGEN MARKERS IN PERSISTENTLY                               INFECTED HUMAN HEP G2 CELL CULTURES                                                         Relative Level of HBV Antigens                                            Conc  % of Untreated Control                                        Compound    (μM) HBsAg    HBeAg  Virion DNA                                ______________________________________                                        Untreated Control                                                                         --      103      110    89                                                            97       90     106                                                           96       108    109                                       Positive Control                                                                          10      100      101    2                                         Dideoxycytidine     112      115    2                                         (2'-3' ddC)         102      98     2                                         Ethanol Control                                                                           --      84       85     102                                       @ 1%                105      93     97                                        Alkyl Lipid 10      78       23     0                                         (CP-51)             114      20     1                                         Alkyl lipid 10      116      33     1                                         (CP-49)             113      26     1                                         Alkyl Lipid 10      68       53     13                                        (CP-9)              64       57     16                                        Platelet Activating                                                                       10      78       45     13                                        Factor (lyso PAF)   80       50     15                                        Nonphosphorus                                                                             10      79       46     9                                         Alkyl Lipid         69       40     10                                        Inverse Choline                                                               (CP-7)                                                                        Lipid-AZT   10      65       35     10                                        Conjugate           69       42     14                                        (CP-126)                                                                      ______________________________________                                    

In contrast, the active antiviral compounds (CP-5 1, CP-49, CP-9, lysoPAF, CP-7, and CP-126) all caused a reduction in Hepatitis B virus eantigen (HBeAg) and virion DNA levels. The most active compounds insuppressing HBeAg and virion DNA levels were CP-5 1 and CP-49. Thesedata suggest that the anti-HBV alkyl lipids and alkyl lipid-AZTconjugate act in a similar manner.

Further analyses were carried out with compound CP-5 1. Again, the cellswere treated for nine days at 37° C. The overlay medium was harvested tomeasure levels of HBsAg, HBeAg, HBcAg, virion DNA, RI DNA and RNAlevels. HBsAg and HBeAg were assayed by EIA (Abbott Laboratories). HBcAgwas assayed by a solid phase EIA. HBV RNA was analyzed by Northern blothybridization using a³² P-labelled 3.2 Kb genomic probe. IntracellularHBV RNA and RI HBV DNA was quantitated using a AMBIS beta scanner. Theresults are given in Table 8.

                                      TABLE 8                                     __________________________________________________________________________    EFFECT OF ALKYL LIPID ON HBV INDUCTION                                        OF ANTIGEN MARKERS, DNA AND RNA IN PERSISTENTLY                               INFECTED HUMAN HEP-G2 CELL CULTURES                                           Relative Level of Antigen Markers DNA and RNA                                                          Intra-                                                                        cellular                                                   Conc           Virion                                                                            HBV-                                                                              RI 3.2 Kb                                                                            Total                                                                            3.6 Kb                                 Compound                                                                            (μM)                                                                          HBsAg                                                                             HBeAg                                                                             HBcAg                                                                             DNA DNA DNA                                                                              RNA RNA                                                                              RNA                                    __________________________________________________________________________    Untreated                                                                           -- 100 100 100 100 100 100                                                                              100 100                                                                              100                                    Controls                                                                      Positive                                                                            10 96  95  102 2    6  46  92 92 105                                    Control                                                                       Dideoxy-                                                                      cytidine (2'-                                                                 3'-ddC)                                                                       Alkyl Lipid                                                                         10 88  24   32 2    12 42 112 84 112                                    (CP-51)                                                                       __________________________________________________________________________

Results of these analyses indicated that CP-51 did not significantlyalter the level or size of HBV induced RNA transcripts of HBsAg. Theseresults suggest that CP-51 does not affect HBV transcription orproduction of HBsAg. In addition to CP-5 1 inhibiting RI HBV-DNA andvirion DNA formation, as seen in Tables 6 and 8, the alkyl lipidsignificantly inhibited the levels of Hepatitis B core antigen (HBcAg),as seen in Table 8. Because both HBeAg and HBcAg are inhibited by CP-51, it appears that the mechanism of action of the compound involvessuppression of nucleocapsid and HBV pregenomic RNA packaging to forminfectious HBV particles. HBeAg is a known proteolytic product of HBcAg,which would account for inhibition of these HBV antigens by CP-51.Inhibition of these essential viral antigens would cause a downstreameffect on the inhibition of intracellular RI HBV DNA and formation ofpackaged virion DNA.

EXAMPLE 8 In Vivo Evaluation of CP-51 for Cytotoxicity andBioavailability

A. Biologic Activity of CP-51

The biologic activity of alkyl lipid CP-5 1 was evaluated in vivo.Sprague Dawley rats were fasted for 18 hours and received a single oraldose of 15 mg/kg of the alkyl lipid CP-5 1 by gavage. An in-dwellingcarotid catheter was used to harvest whole blood at various timespost-dose. The plasma containing the alkyl lipid was diluted 1:2 to1:128 in phosphate buffered saline and assayed for biologic activityusing the HIV-1 syncytial plaque test. CEM-SS cell monolayers wereinfected with HIV-1 and overlaid with RPMI-1640 medium containing serialdilutions of plasma. After incubation for five days at 37° C., thenumber of HIV-1 syncytial plaques was counted as previously described(Kucera et al., AIDS Res. Human Retroviruses, 6: 491-501, (1990)). Thedata in FIG. 1 indicate that CP-51 is rapidly absorbed into the blood ofrats following a single oral dose of 15 mg/kg by gavage. The data alsoindicate that biologically active concentrations of the compound CP-51were obtained in the blood (plasma).

B. Cytotoxicity Assay

CP-51 and AZT were assayed for cytotoxicity by inhibition of mouseprecursor erythrocyte (CFU-E) and granulocyte/macrophage (CFU-GM) cellcolony formation. Mouse bone marrow precursor cells were suspended insoft-agar-RPMI-1640 medium supplmented with cell growth factors andserial concentrations of alkyl lipid. After incubation at 37° C. toallow cell colony formation, the number of colony-forming units in thepresence and absence of alkyl lipid was calculated to determine theinhibitory concentration for 50% (IC₅₀). The inhibitory concentration50% for colony formation (IC₅₀) for CP-51 was 41 microgram/ml (CFU-E)and II microgram/ml (CFU-GM) compared to 0.43 microgram/ml (CFU-E) and0.27 microgram/ml (CFU-GM) for AZT. These results indicated that CP-51was significantly less cytotoxic than AZT.

C. Inhibition of Retrovirus-Induced Splenomegaly

Mice were infected I.P. with Friend-Leukemia virus and not treated ortreated I.P. with daily doses of phospholipid. After 14 days infectionand treatment, the mice were sacrificed, the spleens were removed andweighed to determine the percentage of control splenomegaly. Asindicated in Table 9, CP-51 inhibited retrovirus-induced splenomegaly43% in mice receiving a dose of 12.5 mg/kg/day intra peritoneallycompared to 10% inhibition by the same dose of AZT. Even a single doseof 25 mg/kg CP-51 inhibited splenomegaly by 36%. These data indicatethat CP-51 has anti-retrovirus activity in infected animals.

                  TABLE 9                                                         ______________________________________                                        Effect of Alkyl Lipid in Retrovirus                                           (Friend-Leukemia Virus) Infected Mice                                                             Spleen Wt (g) % of Control                                Compound                                                                              Dose (mg/kg/day)                                                                          (day + 14)    Splenomegaly                                ______________________________________                                        Virus   --          2.091 ± 0.113                                                                         (4).sup.a                                                                          100                                       Control                                                                       CP-51   12.5.sup.b  0.891.sup.c ± 0.393                                                                   (4)  43                                                25.sup.d    0.761.sup.c ± 0.358                                                                   (4)  36                                        AZT     12.5.sup.b  0.219.sup.e + 0.028                                                                      (5)  10                                        ______________________________________                                         .sup.a Number of animals tested                                               .sup.b Daily treatment, day 0 (+1 hour) - day + 13; sacrifice day + 14        .sup.c p ≦ 0.05 Utest                                                  .sup.d One treatment, day 0 (+1 hour); sacrifice day + 14                     .sup.e p ≦ 0.01 Utest                                             

All patents, patent documents and publications are incorporated byreference herein. The foregoing examples are illustrative of the presentinvention, and are not to be construed as limiting thereof. Theinvention is defined by the following claims, with equivalents of theclaims to be included therein.

That which is claimed is:
 1. A method of treating a hepatitis B virusinfection comprising administering to a human subject in need of suchtreatment an effective hepatitis virus-combatting amount of a compoundof Formula I; ##STR7## wherein Y is S, O, NH, NCH₃, NHC(O), or NCH₃C(O);R₁ is unbranched or branched, saturated or unsaturated C 14-C18alkyl, alkenyl, or alkynyl; X is a covalent bond or methylene optionallysubstituted with hydroxyl, C1-C20 alkyl, C1-C20 alkoxy, C1-C20 alkylthioor C1-C20 alkylamido; and D is selected from the group consisting ofmoieties of Formula V or Formula VI; wherein Formula V is

    -(PO.sub.4).sup.13 -E                                      (V)

wherein E is -J-N⁺ (R₂)(R₃)(R₄), wherein J is C1 -C4 alkyl optionallysubstituted one to three times with methyl or ethyl; and R₂, R₃, and R₄are independently selected from the group consisting of H and C1-C3alkyl; or a pharmaceutical salt thereof.
 2. A method according to claim1, wherein Y is NHC(O).
 3. A method according to claim 1, wherein X ismethylene substituted with hydroxyl or C1-C4 alkoxy.
 4. A methodaccording to claim 1, wherein X is methylene substituted with hydroxylor C1-C4 alkoxy, Y is NHC(O), and R₁ is C14-C18 alkyl.
 5. A methodaccording to claim 1, wherein X is methylene substituted with hydroxylor C1-C4 alkoxy, Y is S, and R₁ is C14-C18 alkyl.
 6. A method accordingto claim 1, wherein X is a covalent bond, Y is S, and R₁ is C14-C18alkyl.
 7. A method of treating a hepatitis B viral infection comprisingadministering a human subject in need of such treatment an effectivehepatitis B virus-combatting amount of a compound of Formula II:##STR8## wherein Y is S, O, NH, NCH₃, NHC(O), or NCH₃ C(O); R₁ is anunbranched or branched, saturated or unsaturated C14-C18 alkyl, alkenyl,or alkynyl; X is a covalent bond or methylene optionally substitutedwith hydroxyl, C1-C20 alkyl, C1-C20 alkoxy, C1-C20 alkylthio or C1-C20alkylamido; J is C1-C4 alkyl optionally substituted one to three timeswith methyl or ethyl; and R₂, R₃, and R₄ are independently selected fromthe group consisting of H and C1-C3 alkyl; or a pharmaceutical saltthereof.
 8. A method according to claim 7, wherein J is ethylene, andwherein R₂, R₃, and R₄ are each methyl.
 9. A method according to claim7, wherein Y is NHC(O).
 10. A method according to claim 7, wherein X ismethylene substituted with hydroxyl or C1-C4 alkoxy.
 11. A methodaccording to claim 7, wherein X is methylene substituted with hydroxylor C1-C4 alkoxy, Y is NHC(O), and R₁ is C14-C18 alkyl.
 12. A methodaccording to claim 7, wherein X is methylene substituted with hydroxylor C1-C4 alkoxy, Y is S, and R₁ is C14-C18 alkyl.
 13. A method accordingto claim 7, wherein X is a covalent bond, Y is S, and R₁ is C14-C18alkyl.
 14. A method according to claim 7, wherein the hepatitiscombatting compound of Formula II is selected from the group consistingof rac-3-octadecanamido-2-ethyoxy-1-propylphosphocholine,rac-3-hexadecanamido-2-ethoxy-1-propylphosphocholine,2-hexadecylthio-1-ethylphosphocholine, andrac-3-octadecyloxy-2-hydroxy-1-propyl phosphocholine.